The present invention relates generally to a new cytokine having diagnostic and therapeutic uses. In particular, the present invention relates to a novel interferon, designated xe2x80x9cinterferon-xcex5,xe2x80x9d and to nucleic acid molecules encoding interferon-xcex5.
Cellular differentiation of multicellular organisms is controlled by hormones and polypeptide growth factors. These diffusable molecules allow cells to communicate with each other and act in concert to form tissues and organs, and to repair and regenerate damaged tissue. Examples of hormones and growth factors include the steroid hormones, parathyroid hormone, follicle stimulating hormone, the interferons, the interleukins, platelet derived growth factor, epidermal growth factor, and granulocyte-macrophage colony stimulating factor, among others.
Hormones and growth factors influence cellular metabolism by binding to receptor proteins. Certain receptors are integral membrane proteins that bind with the hormone or growth factor outside the cell, and that are linked to signaling pathways within the cell, such as second messenger systems. Other classes of receptors are soluble intracellular molecules.
Of particular interest, from a therapeutic standpoint, are the interferons (reviews on interferons are provided by De Maeyer and De Maeyer-Guignard, xe2x80x9cInterferons,xe2x80x9d in The Cytokine Handbook, 3rd Edition, Thompson (ed.), pages 491-516 (Academic Press Ltd. 1998), and by Walsh, Biopharmaceuticals: Biochemistry and Biotechnology, pages 158-188 (John Wiley and Sons 1998)). Interferons exhibit a variety of biological activities, and are useful for the treatment of certain autoimmune diseases, particular cancers, and the enhancement of the immune response against infectious agents, including viruses, bacteria, fungi, and protozoa. To date, six forms of interferon have been identified, which have been classified into two major groups. The so-called xe2x80x9ctype Ixe2x80x9d interferons include interferon-xcex1, interferon-xcex2, interferon-xcfx89, interferon-xcex4, and interferon-xcfx84. Currently, interferon-xcex3 and one subclass of interferon-xcex1 are the only type II interferons.
Type I interferons are thought to be derived from the same ancestral gene, and the type I interferons have retained sufficient similar structure to act by the same cell surface receptor. The xcex1-chain of the human interferon-xcex1/xcex2 receptor comprises an extracellular N-terminal domain, which has the characteristics of a class II cytokine receptor. Interferon-xcex3 does not share significant homology with the type I interfetons or with the type II interferon-xcex1 subtype, but shares a number of biological activities with the type I interferons.
In humans, at least 16 non-allelic genes code for different subtypes of interferon-xcex1, while interferons xcex2 and xcfx89 are encoded by single genes. Type I interferon genes are clustered in the short arm of chromosome 9. Unlike typical structural human genes, interferon-xcex1, interferon-xcex2, and interferon-xcfx89 lack introns. A single gene for human interferon-xcex3 is localized on chromosome 12 and contains three introns. To date, interferon-xcfx84 has been described only in cattle and sheep, while interferon-xcex4 has been described only in pigs.
It is believed that all cells may be capable of producing interferons a and xcex2 in response to viral infection, double-stranded RNA molecules, growth factors, and cytokines. Normally, however, interferon-xcex1 is produced by lymphocytes, macrophages, and monocytes, while interferon-xcex2 is synthesized by fibroblasts and some epithelial cells. Interferon-xcex3 is produced by T cells or natural killer cells.
Clinicians are taking advantage of. the multiple activities of interferons by using the proteins to treat a wide range of conditions. For example, one form of interferon-xcex1 has been approved for use in more than 50 countries for the treatment of medical conditions such as hairy cell leukemia, renal cell carcinoma, basal cell carcinoma, malignant melanoma, AIDS-related Kaposi""s sarcoma, multiple myeloma, chronic myelogenous leukemia, non-Hodgkin""s lymphoma, laryngeal papillomatosis, mycosis fungoides, condyloma acuminata, chronic hepatitis B, hepatitis C, chronic hepatitis D, and chronic non-A, non-B/C hepatitis. The U.S. Food and Drug Administration has approved the use of interferon-xcex2 to treat multiple sclerosis, a chronic disease of the nervous system. Interferon-xcex3 is used to treat chronic granulomatous diseases, in which the interferon enhances the patient""s immune response to destroy infectious bacterial, fungal, and protozoal pathogens. Clinical studies also indicate that interferon-xcex3 may be useful in the treatment of AIDS, leishmaniasis, and lepromatous leprosy.
Although new uses of known interferons may be discovered, a need exists for the provision of new interferons for biopharmaceuticals.
The present invention provides a novel interferon, designated xe2x80x9cinterferon-xcex5.xe2x80x9d The present invention also provides interferon-xcex5 polypeptides and interferon-xcex5 fusion proteins, as well as nucleic acid molecules encoding such polypeptides and proteins.
In particular, the present invention provides isolated polypeptides having an amino acid sequence that is at least 70% identical to an amino acid sequence selected from the group consisting of (a) amino acid residues 22-208 of SEQ ID NO:2, (b) amino acid residues 22-208 of SEQ ID NO:5, (c) the amino acid sequence of SEQ ID NO:2, and (d) the amino acid sequence of SEQ ID NO:5, wherein the isolated polypeptide either specifically binds with an antibody that specifically binds with a polypeptide having the amino acid sequence of either SEQ ID NO:2 or SEQ ID NO:5, or exhibits anti-viral activity or anti-prolifertive activity. The present invention also provides isolated polypeptides having an amino acid sequence that is at least 80%, or at least 90%, identical to one of amino acid sequences (a)-(d). Examples of such polypeptides include polypeptides comprising the amino acid sequence of either SEQ ID NO:2 or SEQ ID NO:5. The present invention also contemplates isolated polypeptides comprising a first amino acid sequence consisting of amino acid residues 22 to 208 of either SEQ ID NO:2 or SEQ ID NO:5, as well as polypeptides that further comprise a signal secretory sequence that resides in an amino-terminal position relative to the first amino acid sequence, wherein the signal secretory sequence comprises amino acid residues 1 to 21 of the amino acid sequence of SEQ ID NO:2. The present invention also includes various truncated variant interferon-xcex5 polypeptides, as described below.
The present invention further provides pharmaceutical compositions that comprise such polypeptides, and a pharmaceutically acceptable carrier.
The present invention also includes variant human interferon-xcex5 polypeptides, wherein the amino acid sequence of the variant is characterized by at least one amino acid substitution within SEQ ID NO:2 selected from the group consisting of: (a) an alanine residue for Thr77, (b) a threonine residue for Ser38, (c) a valine residue for Ile80, (d) an aspartate residue for Glu107, and (e) a valine residue for Ile167. Additional variant human interferon-xcex5 polypeptides include those in which the amino acid sequence of the variant polypeptide shares an identity with the amino acid sequence of SEQ ID NO:2 selected from the group consisting of at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, or greater than 95% identity, and wherein any difference between the amino acid sequence of the variant polypeptide and the amino acid sequence of SEQ ID NO:2 is due to one or more conservative amino acid substitutions.
The present invention also includes human interferon polypeptides that comprise the amino acid sequence motif xe2x80x9c[LFI][HY]E[VML][IML]Q[QN][TISV]F[NSA][LI]FRxe2x80x9d (SEQ ID NO:30), wherein the motif is further defined by at least one condition selected from the group consisting of: (a) the first residue is L, (b) the second residue is H, (c) the seventh residue is Q, (d) the tenth residue is S, and (e) the eleventh residue is L. The present invention further provides human interferon polypeptides that comprise the amino acid sequence motif xe2x80x9cYS[PSDTH]CAW[EAT][VTI]V[RQ][AMLV]EIxe2x80x9d (SEQ ID NO:31), wherein the motif is further defined by at least one condition selected from the group consisting of. (a) the third residue is T, (b) the seventh residue is A, and (c) the tenth residue is Q.
The present invention further provides human interferon polypeptides that comprise an amino acid sequence consisting of amino acid residues 189 to 208 of SEQ ID NO:2. Suitable polypeptides include those in which the amino acid sequence occurs at the C-terminus of the polypeptide.
The present invention also contemplates polypeptides consisting of amino acid residues 22 to 94 of either SEQ ID NO:2 or SEQ ID NO:5.
The present invention also provides isolated nucleic acid molecules that encode an interferon-xcex5 polypeptide, wherein the nucleic acid molecule is selected from the group consisting of (a) a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:3, (b) a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:6, (c) a nucleic acid molecule that remains hybridized following stringent wash conditions to a nucleic acid molecule consisting of the nucleotide sequence of SEQ ID NO:1, or the complement of SEQ ID NO:1, and (d) a nucleic acid molecule that remains hybridized following stringent wash conditions to a nucleic acid molecule consisting of the nucleotide sequence of SEQ ID NO:4, or the complement of SEQ ID NO:4. Such nucleic acid molecules include those in which any difference between the amino acid sequence encoded by the nucleic acid molecule and the corresponding amino acid sequence of either SEQ ID NO:2 or SEQ ID NO:5 is dueto a conservative amino acid substitution.
The present invention further provides isolated nucleic acid molecules, comprising the nucleotide sequence of nucleotides 517 to 1077 of either SEQ ID NO:1 or SEQ ID NO:4, isolated nucleic acid molecules comprising the nucleotide sequence of nucleotides 454 to 1077 of either SEQ ID NO:1 or SEQ ID NO:4, and isolated nucleic acid molecules comprising the nucleotide sequence of nucleotides 1 to 1234 of either SEQ ID NO:1 or SEQ ID NO:4.
The present invention also includes murine interferon-xcex5, such as isolated polypeptides comprising a first amino acid sequence consisting of amino acid residues 22 to 192 of SEQ ID NO:24. These polypeptides can further comprise a signal secretory sequence that resides in an amino-terminal position relative to the first amino acid sequence, wherein the signal secretory sequence comprises amino acid residues 1 to 21 of the amino acid sequence of SEQ ID NO:24. Examples of variant murine interferon-xcex5 molecules include isolated polypeptides having an amino acid sequence that is at least 70%, at least 80%, or at least 90% identical to either amino acid residues 22-192 of SEQ ID NO:24, or the amino acid sequence of SEQ ID NO:24, wherein the isolated polypeptide either (a) specifically binds with an antibody that specifically binds with a polypeptide consisting of the amino acid sequence of SEQ ID NO:24, or (b) exhibits anti-viral activity or anti-proliferative activity. Additional variant murine interferon-xcex5 polypeptides include those in which the amino acid sequence of the variant polypeptide shares an identity with the amino acid sequence of SEQ ID NO:24 selected from the group consisting of at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, or greater than 95% identity, and wherein any difference between the amino acid sequence of the variant polypeptide and the amino acid sequence of SEQ ID NO:24 is due to one or more conservative amino acid substitutions.
The present invention further contemplates isolated nucleic acid molecules that encode an interferon-xcex5 polypeptide, wherein the nucleic acid molecule is selected from the group consisting of (a) a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:25, and (b) a nucleic acid molecule that remains hybridized following stringent wash conditions to a nucleic acid molecule consisting of the nucleotide sequence of SEQ ID NO:23, or the complement of SEQ ID NO:23. Such nucleic acid molecules include those in which any difference between the amino acid sequence encoded by the nucleic acid molecule and the corresponding amino acid sequence of SEQ ID NO:24 is due to a conservative amino acid substitution. An illustrative nucleic acid molecule that encodes murine interferon-xcex5 comprises the nucleotide sequence of nucleotides 842 to 1354 of SEQ ID NO:23.
The present invention further provides chimeric interferon-xcex5 proteins having the structure: [hA or mA]-[hAB or mAB]-[hB or mB]-[hBC or mBC]-[hC or mC]-[hCD or mCD]-[hD or mD]-[hDE or mDE]-[hE or, mE], wherein the designations xe2x80x9cAxe2x80x9d through xe2x80x9cExe2x80x9d indicate an interferon-xcex5 helix region, designations xe2x80x9cABxe2x80x9d trough xe2x80x9cDExe2x80x9d indicate an interferon-xcex5 loop region, and xe2x80x9chxe2x80x9d and xe2x80x9cmxe2x80x9d refer to human interferon-xcex5 and murine interferon-xcex5, respectively. Illustrative chimeric interferon-xcex5 proteins include polypeptides having the structure [mA]-[hAB]-[hB]-[hBC]-[hC]-[mCD]-[mD]-[hDE]-[mE], [mA]-[hAB]-[hB]-[hBC]-[mC]-[hCD]-[hD]-[mDE]-[hE], [mA]-[hAB]-[hB]-[hBC]-[mC]-[hCD]-[mD]-[mDE]-[hE], and [hA]-[mAB]-[mB]-[mBC]-[mC]-[hCD]-[hD]-[mDE]-[hE]. Such chimeric interferon-xcex5 proteins can further comprise at least one of (a) a signal sequence, wherein the signal sequence is located in an N-terminal position, and (b) a human interferon-xcex5 C-terminal amino acid sequence or a murine interferon-xcex5 C-terminal amino acid sequence. An illustrative human interferon-xcex5 C-terminal amino acid sequence comprises amino acid residues 185 to 208 of SEQ ID NO:2, and an illustrative murine interferon-xcex5 C-terminal amino acid sequence comprises amino acid residues 184 to 192 of SEQ ID NO:24. The present invention also includes nucleic acid molecules encoding such chimeric interferon-xcex5 proteins.
The present invention also provides vectors and expression vectors comprising such nucleic acid molecules, recombinant host cells comprising such vectors and expression vectors, and recombinant viruses comprising such expression vectors. These expression vectors and recombinant host cells can be used to prepare interferon-xcex5 polypeptides. In addition, the present invention provides pharmaceutical compositions, comprising a pharmaceutically acceptable carrier and at least one of such an expression vector or recombinant virus. Preferably, such pharmaceutical compositions comprise a human interferon-xcex5 gene, a murine interferon-xcex5 gene, or a variant thereof.
The present invention further contemplates antibodies and antibody fragments that specifically bind with interferon-xcex5 polypeptides. Such antibodies include polyclonal antibodies, murine monoclonal antibodies, humanized antibodies derived from murine monoclonal antibodies, and human monoclonal antibodies. Examples of antibody fragments include F(abxe2x80x2)2, F(ab)2, Fabxe2x80x2, Fab, Fv, scFv, and minimal recognition units.
The present invention also provides methods for detecting the presence of interferon-xcex5 RNA in a biological sample, comprising the steps of:
(a) contacting an interferon-xcex5 nucleic acid probe under hybridizing conditions with either (i) test RNA molecules isolated from the biological sample, or (ii) nucleic acid molecules synthesized from the isolated RNA molecules, wherein the probe has a nucleotide sequence comprising a portion of the nucleotide sequence selected from the group consisting of SEQ ID NO:1, the complement of SEQ ID NO:1, SEQ ID NO:4, and the complement of SEQ ID NO:4, and
(b) detecting the formation of hybrids of the nucleic acid probe and either the test RNA molecules or the synthesized nucleic acid molecules,
wherein the presence of the hybrids indicates the presence of interferon-xcex5 RNA in the biological sample.
In addition, the presence of interferon-xcex5 polypeptide in a biological sample can be detected by methods that comprise the steps of (a) contacting the biological sample with an antibody, or an antibody fragment, that specifically binds with a polypeptide having the amino acid sequence of either SEQ ID NO:2 or SEQ ID NO:5, wherein the contacting is performed under conditions that allow the binding of the antibody or antibody fragment to the biological sample, and (b) detecting any of the bound antibody or bound antibody fragment.
The present invention also provides kits for detecting interferon-xcex5 nucleic acid molecules or interferon-xcex5 polypeptides. For example, a kit for detection of interferon-xcex5 nucleic acid molecules may comprise a container that comprises a nucleic acid molecule, wherein the nucleic acid molecule is selected from the group consisting of (a) a nucleic acid molecule comprising the nucleotide sequence of nucleotides 517 to 1077 of SEQ ID NO:1, (b) a nucleic acid molecule comprising the complement of the nucleotide sequence of SEQ ID NO:1, (c) a nucleic acid molecule that is a fragment of (a) consisting of at least eight nucleotides, (d) a nucleic acid molecule that is a fragment of (b) consisting of at least eight nucleotides, (e) a nucleic acid molecule comprising the nucleotide sequence of nucleotides 517 to 1077 of SEQ ID NO:4, (f) a nucleic acid molecule comprising the complement of the nucleotide sequence of SEQ ID NO:4, (g) a nucleic acid molecule that is a fragment of (e) consisting of at least eight nucleotides, and (h) a nucleic acid molecule that is a fragment of (f) consisting of at least eight nucleotides. Such kits may further comprise a second container that comprises one or more reagents capable of indicating the presence of the nucleic acid molecule. A kit for detection of interferon-xcex5 polypeptide may comprise a container that comprises an antibody, or an antibody fragment, that specifically binds with a polypeptide having the amino acid sequence of either SEQ ID NO:2 or SEQ ID NO:5.
The present invention also contemplates isolated nucleic acid molecules comprising a nucleotide sequence that encodes an interferon-xcex5 secretion signal sequence and a nucleotide sequence that encodes a biologically active polypeptide, wherein the interferon-xcex5 secretion signal sequence comprises an amino acid sequence of residues 1 to 21 of SEQ ID NO:2. Illustrative biologically active polypeptides include Factor VIIa, proinsulin, insulin, follicle stimulating hormone, tissue type plasminogen activator, tumor necrosis factor, interleukin, colony stimulating factor, interferon, erythropoietin, and thrombopoietin. Moreover, the present invention provides fusion proteins comprising an interferon-xcex5 secretion signal sequence and a polypeptide, wherein the interferon-xcex5 secretion signal sequence comprises an amino acid sequence of residues 1 to 21 of SEQ ID NO:2. Additional fusion proteins comprise an interferon-xcex5 moiety and an immunoglobulin moiety. An illustrative immunoglobulin moiety is an immunoglobulin heavy chain constant region, such as a human Fc fragment The present invention also includes isolated nucleic acid molecules that encode such fusion proteins.
The present invention also contemplates anti-idiotype antibodies, or anti-idiotype antibody fragments, that specifically bind with an anti-interferon-xcex5 antibody or antibody fragment, wherein the anti-idiotype antibody, or anti-idiotype antibody fragment, possesses anti-viral activity or anti-proliferative activity.
The present invention further provides: (1) isolated polypeptides having an amino acid sequence consisting of amino acid residues 27-94 of either SEQ ID NO:2 or SEQ ID NO:5, (2) isolated polypeptides that comprise a peptide segment having amino acid residues 173 to 188 of SEQ ID NO:2, and (3) isolated polypeptides that comprise a first peptide segment having amino acid residues 173 to 188 of SEQ ID NO:2 and a second peptide segment that resides in a carboxyl-terminal position relative to the first peptide segment, wherein the second peptide segment has the amino acid sequence of amino acid residues 185 to 208 of SEQ ID NO:2. The present invention also includes isolated nucleic acid molecules that encode these polypeptides.
The present invention includes methods for detecting the presence of murine interferon-xcex5 RNA in a biological sample, comprising the steps of: (a) contacting an interferon-xcex5 nucleic acid probe under hybridizing conditions with either (i) test RNA molecules isolated from the biological sample, or (ii) nucleic acid molecules synthesized from the isolated RNA molecules, wherein the probe has a nucleotide sequence comprising a portion of the nucleotide sequence of either SEQ ID NO:23, or the complement of SEQ ID NO:23, and (b) detecting the formation of hybrids of the nucleic acid probe and either the test RNA molecules or the synthesized nucleic acid molecules, wherein the presence of the hybrids indicates the presence of interferon-xcex5 RNA in the biological sample. The present invention also provides methods for detecting the presence of murine interferon-xcex5 in a biological sample, comprising the steps of: (a) contacting the biological sample with an antibody, or an antibody fragment, that specifically binds with a polypeptide consisting of the amino acid sequence of SEQ ID NO:24, wherein the contacting is performed under conditions that allow the binding of the antibody or antibody fragment to the biological sample, and (b) detecting any of the bound antibody or bound antibody fragment. The present invention also contemplates kits for performing these methods.
The present invention also provides expression vectors comprising a murine interferon-xcex5 promoter, wherein the promoter comprises nucleotides 1 to 778 of SEQ ID NO:23.
The present invention further includes methods for detecting an alteration in chromosome 9. In particular, human interferon-xcex5 nucleotide sequences can be used to examine chromosome 9p, for example, in the 9p22.2 region. Illustrative chromosomal aberrations at the interferon-xcex5 gene locus include aneuploidy, gene copy number changes, insertions, deletions, restriction site changes and rearrangements. These aberrations can occur within flanking sequences, including upstream promoter and regulatory regions, and can be manifested as physical alterations within a coding sequence or changes in gene expression level. Such methods are effected by examining the interferon-xcex5 gene and its gene products. In general, suitable assay methods include molecular genetic techniques known to those in the art, such as restriction fragment length polymorphism analysis, short tandem repeat analysis employing polymerase chain reaction techniques, ligation chain reaction, ribonuclease protection assays, use of single-nucleotide polymorphisms, protein truncation assays, and other genetic linkage techniques known in the art.
In particular, the present invention provides methods for diagnosing an alteration in the interferon-xcex5 gene of an individual, comprising: (a) amplifying nucleic acid molecules that encode interferon-xcex5 from RNA isolated from a biological sample of the individual, and (b) detecting a mutation in the amplified nucleic acid molecules, wherein the presence of a mutation indicates an alteration in the interferon-xcex5 gene. Similarly, methods of detecting a chromosome 9p22.2 abnormality in a subject comprise: (a) amplifying nucleic acid molecules that encode interferon-xcex5 from RNA isolated from a biological sample of the subject, and (b) detecting a mutation in the amplified nucleic acid molecules, wherein the presence of a mutation indicates a chromosome 9p22.2 abnormality. In variations of these methods, the detecting step is performed by comparing the nucleotide sequence of the amplified nucleic acid molecules to the nucleotide sequence of SEQ ID NOs:1 or 4. Alternatively, the detecting step can be performed by fractionating the amplified nucleic acid molecules and control nucleic acid molecules that encode the amino acid sequence of SEQ ID NOs:2 or 5, and comparing the lengths of the fractionated amplified and control nucleic acid molecules. Exemplary methods for amplification include polymerase chain reaction or reverse transcriptase-polymerase chain reaction.
The present invention also includes methods for detecting a chromosome 9p22.2 abnormality in a subject comprising: (a) amplifying nucleic acid molecules that encode interferon-xcex5 from RNA isolated from a biological sample of the subject, (b) transcribing the amplified nucleic acid molecules to express interferon-xcex5 mRNA, (c) translating interferon-xcex5 mRNA to produce interferon-xcex5 polypeptides, and (d) detecting a mutation in the interferon-xcex5 polypeptides, wherein the presence of a mutation indicates a chromosome 9p22.2 abnormality. In variations of these methods, the detection step can be performed by fractionating, under denaturing conditions, the interferon-xcex5 polypeptides and control polypeptides that encode the amino acid sequence of SEQ ID NOs:2 or 5, and comparing the sizes of the fractionated amplified and control polypeptides. Similar methods can be used to detect a mutation of an interferon-xcex5 gene in an individual.
The present invention further includes methods of inhibiting a viral infection of cells, comprising the step of administering a composition comprising interferon-xcex5 to the cells. For example, the composition can be a pharmaceutical composition, which is administered in a therapeutically effective amount to a subject which has a viral infection. In vivo treatment of a viral infection can provide at least one physiological effect selected from the group consisting of decreased viral titer, decreased detectable viral antigen, and increased anti-viral antibody titer.
The present invention also includes methods of inhibiting the proliferation of tumor cells, comprising the step of administering a composition comprising interferon-xcex5 to the tumor cells. In an in vivo approach, the composition is a pharmaceutical composition, administered in a therapeutically effective amount to a subject which has a tumor. Such in vivo administration can provide at least one physiological effect selected from the group consisting of decreased number of tumor cells, decreased metastasis, decreased size of a solid tumor, and increased necrosis of a tumor.
The present invention further includes methods of treating a lymphoproliferative disorder in a subject, comprising the step of administering a therapeutically effective amount of pharmaceutical composition to the subject, wherein the pharmaceutical composition comprises interferon-xcex5. Illustrative lymphoproliferative disorders include B-cell lymphoma, chronic lymphatic leukemia, and acute lymphatic leukemia
Methods of treatment can be performed with interferon-xcex5 polypeptides and variants, including human interferon-xcex5, murine interferon-xcex5, chimeric interferon-xcex5, and the like.
These and other aspects of the invention will become evident upon reference to the following detailed description and the attached drawings. In addition, various references are identified below and are incorporated by reference in their entirety.